Factors affecting chloride conductance in apical membrane vesicles from human placenta

Abstract

Apical membrane vesicles from human term placenta were isolated using a magnesium precipitation technique, and the purity of the vesicles was assessed morphologically using scanning and transmission electron microscopy, and biochemically, using marker enzymes. The vesicles were found to be morphologically intact and significantly enriched in enzymes associated with apical membranes. 36Cl- uptake into these vesicles was studied in the presence of an outwardly directed Cl- gradient. This uptake was found to be time dependent, with an initial rapid uptake tending to peak between 10 and 20 min and thereafter decline. Uptake was found to be voltage dependent since 5 microM valinomycin caused a decrease in uptake. The effects of N-phenylanthranilic acid (NPA) and 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) and bumetanide on the initial rate of Cl- were examined in the presence and absence of 5 microM valinomycin. NPA and DIDS inhibited isotope uptake strongly with IC50 values of 0.83 +/- 0.35 microM and 3.43 +/- 0.37 microM, respectively, in the absence of valinomycin. Although valinomycin reduced 36Cl- uptake by about 80% when added before the isotope, DIDS reduced the uptake which remained in a concentration-dependent fashion with an IC50 of 5.6 +/- 2.1 microM. Under these conditions, NPA was without effect at concentrations below 100 microM. Bumetanide was without effect at the concentrations used in the absence of valinomycin. However, following valinomycin pretreatment, bumetanide reduced 36Cl- uptake significantly at 100 microM concentration. Vesicle diameter, as assessed by flow cytometry, did not change under the conditions employed. The effects of some fatty acids were also investigated. Arachidonic acid and linoleic acid inhibited Cl- uptake with IC50 values of 37.6 +/- 14.9 microM and 4.59 +/- 0.51 microM, respectively. Arachidonyl alcohol and elaidic acid were found to be without effect. These studies show that human placental brush border membrane vesicles possess a chloride conductance channel, the activity of which can be measured in the presence of an outwardly directed Cl- gradient and this channel is sensitive to Cl- channel inhibitors, especially N-phenylanthranilic acid, and can be inhibited by unsaturated fatty acids such as arachidonic acid and linoleic acid.

Characterization of Cytosolic Cl^– Channel Inhibitors by Size Exclusion Chromatography

“…Other CP channels were inhibit ed by intra-or extracellular nucleotides and nucleotide analogs [11][12][13][14], Futhermore, cisunsaturated fatty acids, e.g. arachidonic acid, caused inhibition of CP channels [15][16][17], Recently, we have demonstrated that cyto sol from pig kidney, human placenta, HT29 and CFPAC-1 cells inhibits outwardly rectify ing CP channels from respiratory epithelial cells, CFPAC-1 cells, and from HT29 cells [17][18][19], The inhibition showed a rapid onset, was concentration-dependent, reversible, and was caused by a heat-stable factor of unknown structure. Here we report on the fractionation of the cytosol by gel chromatography as a first step to isolate the inhibitory factors.…”

Inhibition by fatty acids of cyclic AMP‐dependent protein kinase activity in brush border membranes isolated from human placental vesicles

“…We would further propose that the enzyme is localized to the cytosolic surface of BBMV prior to lysis since there is evidence for the existence of proteinases on the outer surface of BBMV, which selectively degrade cyclic AMP-stimulated ectokinases in the course of vesicle isolation (De Jonge et al, 1987). AA inhibited PKA activity in our system at a concentration (1O pM) previously shown to inhibit Cl-channel activity in airway epithelial cells (Anderson & Welsh, 1990;Hwang et al, 1990;Faller & Ryan, 1992). A direct role for AA in PKA inhibition is supported by (i) the enhanced effect observed in the presence of inhibitors of metabolism by the lipoxygenase and cyclo-oxygenase pathways (Indo + NDGA) and (ii) by the lack of inhibition in the presence of the AA metabolites PGE2, PGF2., leukotriene C4 or 5-HPETE.…”

“…Brush border membrane vesicles (BBMV) were prepared as described by Faller & Ryan (1992): the central portion between the maternal and foetal surfaces of the placenta was used, following removal of the maternal decidua. The maternal villous tissue was chopped into small pieces, washed in Buffer A (300 mM mannitol, 10 mM HEPES/Tris HCl, pH 7.0), further finely chopped and agitated in 300 ml Buffer A with a magnetic stirrer for 1 h at 4°C.…”

“…to exhibit a Cl-conductance (Dechecchi & Cabrini, 1988;Illsley et al, 1988) which is inhibitable by AA (Faller & Ryan, 1992).…”

Functional transplantation of chloride channels from the human syncytiotrophoblast microvillous membrane to Xenopus oocytes

“…Most of them have been carried out on membrane vesicles prepared from the microvillous (apical) placental trophoblast either by quenching and uptake experiments [9,14,17,33,34] or, less frequently, by patch-clamping giant liposome vesicles [27,36,37]. In addition, human placental ionic channels have been recently reconstituted on planar lipid bilayers [18,19].…”